Water sample pretreatment reagent for detecting total nitrogen in water body, total nitrogen detection kit and detection method

Abstract

The invention discloses a water sample pretreatment reagent for detecting total nitrogen in a water body, a total nitrogen detection kit and a detection method. The water sample pretreatment reagent comprises a reagent A, a reagent B and a reagent C, the reagent A is nitrogen determination alloy; the reagent B is a urease extracting solution; the reagent C is a sodium potassium tartrate tetrahydrate-sodium hydroxide solution; the urease extracting solution is prepared by the following steps: uniformly mixing a stabilizer with glycerol, then adding bean flour, stirring and extracting at normal temperature, taking supernate, filtering and then taking filtrate. The total nitrogen detection kit comprises an ammonia nitrogen detection reagent and a pretreatment reagent. The detection method comprises the following steps: taking a detection water sample, adding the reagent A, uniformly mixing, then adding the reagent B, uniformly mixing, carrying out an enzymatic hydrolysis reaction for 4-6 min, finally adding the reagent C, uniformly mixing, carrying out a reaction to obtain a pretreatment solution, adding the ammonia nitrogen detection reagent into the pretreatment solution, and carrying out detection after color development. The device can measure the sum of urea nitrogen and inorganic total nitrogen in a water body, and is especially suitable for measuring the total nitrogen of swimming pool water and measuring the total nitrogen of related rivers of urea-applied farmland.

Description

Technical Field

[0001] The invention relates to the technical field of total nitrogen detection in water bodies, and in particular to a water sample pretreatment reagent, a total nitrogen detection kit and a detection method for detecting total nitrogen in water bodies containing urea nitrogen and inorganic nitrogen. Background Art

[0002] Total nitrogen in water refers to the sum of nitrogen content in nitrogen compounds such as organic nitrogen, ammonia nitrogen, nitrate nitrogen and nitrite nitrogen. It is internationally recognized as one of the important indicators for measuring the degree of eutrophication of water bodies. The Ministry of Ecology and Environment has listed total nitrogen as the main online measurement factor in surface water and industrial and mining wastewater discharge.

[0003] Methods for determining total nitrogen include ultraviolet spectrophotometry, high temperature oxidation-chemiluminescence, and other improved detection methods. For example, a Chinese invention patent document with publication number CN 115508179 A discloses a method for detecting total nitrogen in water. Organic nitrogen and inorganic nitrogen compounds in a water sample are digested with alkaline potassium persulfate under high temperature and high pressure conditions. Various nitrogen-containing compounds are eventually converted into nitrates in the digestion reaction. The digested water sample is placed in a hydrochloric acid medium, and the reducing agent rapidly reduces and decomposes the nitrates to generate gaseous NO. NO and O3 react to generate excited NO. 2* , when the excited NO 2* When it transitions to the ground state, it emits photons, the intensity of which is proportional to the total nitrogen content in the sample. Therefore, the total nitrogen content in the sample can be determined by measuring the intensity of chemiluminescence.

[0004] Potassium persulfate used in the national standard method or the above patents often has a high nitrogen content, and improper operation during the preparation process may render the reagent ineffective and fail to meet the standard requirements, making it difficult for the laboratory to effectively control its blank background value. The national standard potassium persulfate ultraviolet photometry method requires that the absorbance ratio (A275 / A220)*100% should be less than 20%, the smaller the better, but in actual operation, the above ratio is often greater than 20%, resulting in measurement failure and it is difficult to analyze the cause. In addition, the more complex the water environment, the greater the challenge to accuracy. In addition, the total nitrogen determination using the potassium persulfate method must have a high-temperature digestion step, and the safety of the experimental operation must be paid attention to at all times. Summary of the invention

[0005] The present application provides a water sample pretreatment reagent, a total nitrogen detection kit and a detection method for detecting total nitrogen in water bodies, which can determine the sum of urea nitrogen and inorganic total nitrogen in water bodies, and is particularly suitable for the determination of total nitrogen in swimming pool water and the determination of total nitrogen in relevant rivers of farmland where urea is applied.

[0006] A water sample pretreatment reagent for total nitrogen detection in water, comprising reagent A, reagent B and reagent C; the reagent A is a nitrogen determination alloy; the reagent B is a urease extract; the reagent C is a potassium sodium tartrate-sodium hydroxide solution, in which, by weight percentage, the potassium sodium tartrate concentration is 1%-10%, the sodium hydroxide concentration is 0.1%-10%, and the solvent is water; The preparation of the urease extract comprises: The stabilizer and glycerol are mixed, and then soybean powder is added, stirred at room temperature for 0.5-1.5 hours, and then allowed to stand at 0-5°C for 20-25 hours, and the supernatant is taken and filtered, and the obtained filtrate is the urease extract; The mass ratio of the stabilizer, glycerin and soybean powder is 60-100:5-50:10; The stabilizer, based on 250 mL, contains 1-10 g of anhydrous citric acid, 1-10 g of anhydrous disodium hydrogen phosphate and 1-10 g of EDTA-2Na, the solvent is water, and the pH is 5-7.

[0007] In the pretreatment reagent of the present application, the nitrogen determination alloy converts the inorganic nitrogen (nitrate nitrogen, etc.) in the water sample into ammonium nitrogen, and the urease extract converts the urea nitrogen in the water sample into ammonium nitrogen. The urea nitrogen and inorganic total nitrogen in the water sample are converted into ammonium nitrogen and then reacted with the ammonia nitrogen detection reagent to develop a color, and the sum of urea nitrogen and inorganic total nitrogen in the water body can be determined. In a quantitative water sample, an appropriate amount of nitrogen determination alloy is beneficial to enhancing the activity of urease in the urease extract, and the two cooperate with each other, and the urease extract of the present application can be kept at room temperature for at least 1 year without losing activity.

[0008] Optionally, in the preparation of the urease extract, the extract is stirred at room temperature for 1 hour and then allowed to stand at 0-5°C for 24 hours.

[0009] Optionally, in the preparation of the urease extract, the mass ratio of the stabilizer, glycerol and soybean powder is 80-100:5-15:10.

[0010] Optionally, in the preparation of the urease extract, the stabilizer, based on 250 mL, comprises 1-2 g of anhydrous citric acid, 3-6 g of anhydrous disodium hydrogen phosphate and 5-10 g of EDTA-2Na, the solvent is water, and the pH is 5-7.

[0011] Optionally, in the sodium potassium tartrate-sodium hydroxide solution, by weight percentage, the concentration of sodium potassium tartrate is 3%-5%, the concentration of sodium hydroxide is 0.1%-5%, and the solvent is water.

[0012] Most preferably, The method for preparing urease extract is as follows: Stabilizer preparation: 1g anhydrous citric acid, 5g anhydrous disodium hydrogen phosphate, 7g EDTA-2Na, first dissolve in 200mL pure water, then adjust the pH to 5-7 with sodium hydroxide solution, and add pure water to make up to 250mL; Preparation of soybean powder: grind soybeans with a traditional Chinese medicine grinder, and pass through a 100-mesh sieve for later use; Preparation of urease extract: 90g stabilizer, add 10g glycerol and stir evenly, then add 10g soybean powder and stir for 1h to extract at room temperature, place in the refrigerator and let stand for 24h, take the supernatant and filter it with a 500-mesh sieve, and keep the filtrate for later use.

[0013] In the sodium potassium tartrate-sodium hydroxide solution, by weight percentage, the sodium potassium tartrate concentration is 5%, the sodium hydroxide concentration is 4%, and the solvent is water.

[0014] The present application also provides a water sample pretreatment method for detecting total nitrogen in water using the water sample pretreatment reagent, comprising: Take the test water sample, add the reagent A and mix, then add the reagent B and mix, enzymatically react for 4 to 6 minutes, then add the reagent C, mix and react for 8 to 12 minutes to obtain the pretreatment solution.

[0015] Optionally, based on 1 mL of the test water sample, the added amounts of reagent A, reagent B and reagent C are 30-60 mg, 100-200 uL and 3-5 mL respectively.

[0016] Furthermore, based on 1 mL of the test water sample, the added amounts of the reagent A, reagent B and reagent C are 50 mg, 3 drops (about 150 uL) and 4 mL respectively.

[0017] In a quantitative water sample, in the presence of a urease stabilizer, an appropriate amount of nitrogen alloy has the effect of enhancing the urease activity in the urease extract and catalyzing the reaction; however, when the ratio of the nitrogen alloy and urease is not coordinated, the urea conversion rate may be insufficient or even worse, it may have an inhibitory effect. Therefore, the appropriate reagent addition ratio is also one of the keys to the pretreatment method of this application.

[0018] The present application also provides a detection kit for total nitrogen in water, characterized in that it comprises an ammonia nitrogen detection reagent and the water sample pretreatment reagent.

[0019] Optionally, the ammonia nitrogen detection reagent is an ammonia nitrogen detection reagent with a detection concentration range of 0-20 mg / L.

[0020] The ammonia nitrogen detection reagent itself can be a commercially available product, for example, optionally, the reagent in the ammonia nitrogen colorimetric tube / test pack with a mileage of 0-20 mg / L produced by Zhejiang Luheng Environmental Technology Co., Ltd. can be used. The ammonia nitrogen detection reagent disclosed in the announcement number CN112858271 B can also be used.

[0021] The present application also provides a method for detecting total nitrogen in water using the detection kit, comprising: Take the test water sample, add the reagent A and mix well, then add the reagent B and mix well, carry out enzymatic hydrolysis for 4 to 6 minutes, then add the reagent C, mix well and react for 8 to 12 minutes to obtain the pretreatment solution; The obtained pre-treatment liquid is filtered and the filtrate is taken, diluted in proportion and then added with the ammonia nitrogen detection reagent, and the absorbance value at 620 nm is detected after standing for 4 to 6 minutes; Substitute the obtained absorbance value into the standard curve and calculate the total nitrogen concentration in the test water sample.

[0022] Optionally, based on 1 mL of the test water sample, the added amounts of the reagent A, reagent B and reagent C are 30-60 mg, 100-200 uL and 3-5 mL, respectively. Further, based on 1 mL of the test water sample, the added amounts of the reagent A, reagent B and reagent C are 50 mg, 3 drops (about 150 uL) and 4 mL, respectively.

[0023] The amount of the ammonia nitrogen detection reagent added can be added according to the amount of the ammonia nitrogen detection reagent used in measuring ammonia nitrogen, without any special requirements.

[0024] Compared with the prior art, the present invention has at least one of the following beneficial effects: (1) The current national standard method for determining total nitrogen is potassium persulfate oxidation-ultraviolet spectrophotometry, but the experimental process is relatively cumbersome and the requirements for experimental conditions, reagents, and utensils are also relatively strict. In addition, when measuring actual water samples, especially when ammonia nitrogen accounts for a high proportion of total nitrogen in the water sample, the measured total nitrogen value is often lower than ammonia nitrogen, indicating that this method has certain defects. This method uses ammonia nitrogen detection reagent as the basis to directly convert inorganic nitrogen and urea nitrogen into ammonia nitrogen for determination, which can avoid the above problems.

[0025] (2) This method can be used to determine the sum of urea nitrogen and inorganic total nitrogen in water bodies. It is particularly suitable for the determination of total nitrogen in swimming pool water and related rivers where urea is applied.

[0026] (3) The potassium persulfate oxidation method involves a high-temperature and high-pressure digestion process. This method avoids high-temperature and high-pressure operations and reduces experimental condition restrictions. It can be used as a portable reagent, which is conducive to direct total nitrogen determination in the field.

[0027] (4) The test reagent has good stability, a long shelf life and can be stored at room temperature. The urease extract of the present application can be kept at room temperature for at least 1 year without losing activity, while the urease purchased from the conventional market needs to be stored at -20°C and is difficult to circulate on the market. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the color development result diagram in Example 1; Figure 2 It is the standard curve diagram in Example 1; Figure 3 This is the color development result diagram in Example 5. DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0031] Example 1 Total nitrogen standard stock solution (100 mg / L): Weigh 0.7218 g potassium nitrate (dried at 105-110°C for 2 hours, cooled to room temperature in a desiccator) and dissolve in an appropriate amount of water, transfer to a 1000 mL volumetric flask, dilute to the mark with water, and mix well. Prepare and use immediately. The preparation of total nitrogen standard stock solution refers to the national standard: "Determination of total nitrogen in water quality by alkaline potassium persulfate digestion ultraviolet spectrophotometry" (HJ636-2012).

[0032] The total nitrogen standard solution (100 mg / L) was prepared into a standard gradient (calibration gradient) in the following manner: wherein the standard gradient solution was prepared from high concentration to low concentration.

[0033] Table 1

[0034] (2) Detection reagents Reagent A: nitrogen alloy; Reagent B: Urease extract, prepared as follows: Stabilizer preparation: 1g anhydrous citric acid, 5g anhydrous disodium hydrogen phosphate, 7g EDTA-2Na, first dissolve in 200mL pure water, then adjust the pH to 5-7 with sodium hydroxide solution, and add pure water to make up to 250mL.

[0035] Preparation of soybean powder: Grind the soybeans with a traditional Chinese medicine grinder and pass through a 100-mesh sieve for later use.

[0036] Preparation of urease extract: 90g stabilizer, add 10g glycerol and stir evenly, then add 10g soybean powder and stir for 1h to extract at room temperature, place in the refrigerator and let stand for 24h, take the supernatant and filter it with a 500-mesh sieve, and keep the filtrate for later use.

[0037] Reagent C: potassium sodium tartrate-sodium hydroxide solution (mass percentage concentration), potassium sodium tartrate 5%, sodium hydroxide concentration 4%, solvent is water.

[0038] Ammonia nitrogen detection reagent: The detection range is 0-20mg / L and can be purchased commercially. Here we take the ammonia nitrogen colorimetric tube / test package with a range of 0~20mg / L in the water quality rapid detection series provided by Zhejiang Luheng Environmental Technology Co., Ltd. as an example.

[0039] (3) Measurement steps Take 1 ml of total nitrogen standard solution of different concentrations (0, 1 mg / L, 5 mg / L, 10 mg / L, 25 mg / L, 50 mg / L, 75 mg / L, 100 mg / L), add 50 mg of reagent A, shake well, add 3 drops of reagent B (conventional dropper used in the laboratory, about 150 uL), shake well, enzymatic hydrolysis for 5 minutes, add 4 mL of reagent C and shake well, react for 10 minutes, filter with rapid qualitative filter paper, take 1 mL of filtrate, add 4 mL of pure water, shake well, add the reagent in the whole tube of ammonia nitrogen colorimetric tube, let it react for 5 minutes, and use a multi-parameter water quality analyzer (Multi-parameter Water Quality Detector LH-T725, Zhejiang Luheng Environmental Technology Co., Ltd.) to measure the absorbance value at 620 nm.

[0040] Six parallel tests were performed for each concentration.

[0041] (4) Measurement results The result after adding the reagent in the ammonia nitrogen colorimetric tube is as follows Figure 1 shown.

[0042] The absorbance detection results are shown in Table 2.

[0043] Table 2

[0044] With absorbance as the horizontal axis and total nitrogen concentration (mg / L) as the vertical axis, draw a standard curve as shown below: Figure 2As shown: y = 29.989x-0.7496, R 2 =0.9981, x is the absorbance value, y is the total nitrogen concentration, the unit is mg / L.

[0045] Example 2 Screening of the dosage of reagent A Using the detection reagent and detection method of Example 1, potassium nitrate, a standard substance of total nitrogen, is used as a benchmark to explore the conversion of the amount of reagent A to the nitrogen content of the urea solution, with 90%-110% as the qualified conversion rate. The results are shown in Table 3. From the results of Table 3, it can be seen that when the amount of reagent A is less than 30 mg, the urea nitrogen conversion rate of more than 5 mg / L is less than 85.86%, and the lowest conversion rate is only 71.41%; when the amount of reagent A is higher than 60 mg, the urea nitrogen conversion rate exceeds 110%. Therefore, 30-60 mg is suitable.

[0046] Table 3

[0047] Example 3 Screening of the dosage of reagent B Using the detection reagent and detection method of Example 1, potassium nitrate, a standard substance of total nitrogen, was used as a benchmark to explore the conversion of the amount of reagent B to the nitrogen content of the urea solution, with 90%-110% as the qualified conversion rate. The results are shown in Table 4. From the results in Table 4, it can be seen that when the amount of reagent B is 1 drop (about 50 uL), the urea nitrogen conversion rate is lower than 90.86%, and the lowest conversion rate is only 74.83%; when the amount of reagent B is 5 drops (about 250 uL), the 100mg / L urea nitrogen conversion rate exceeds 126%. Therefore, 2-4 drops (about 100~200 uL) are suitable, and the dropper used is a conventional dropper for laboratory use.

[0048] Table 4

[0049] Example 4 Effect of the order of reagent addition Take 1 ml of urea solution of different concentrations (in terms of nitrogen, the total nitrogen concentration equivalent is 0, 1 mg / L, 5 mg / L, 10 mg / L, 25 mg / L, 50 mg / L, 75 mg / L, 100 mg / L respectively), use the detection reagent of Example 1, compared with the determination steps of Example 1, the difference is that reagent C is added first, and then reagents A and B are added for enzymatic hydrolysis reaction. The results are as follows: Figure 3 As shown, there is no color difference in each concentration gradient.

[0050] Example 5 Actual water sample spike recovery Using the detection reagent and standard curve of Example 1, the same determination steps as Example 1, actual water samples including pure water, tap water, river water and seawater were taken to conduct spike recovery tests. The results are shown in Table 5, and the spike recovery rates are between 91.89% and 108.70%.

[0051] Table 5

[0052] Example 6 Comparison of Anti-interference Capability Compared with two foreign total nitrogen detection reagents, according to the relevant instructions or test results, the anti-interference ability of related ions is equal to or better than that of total nitrogen reagent (1), and the anti-interference ability of related ions except copper ions and aluminum ions is equal to or better than that of total nitrogen reagent (2). The results are shown in Table 6, and the blanks in the following table are unknown items.

[0053] Table 6

[0054] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims.

Claims

1. A water sample pretreatment reagent for total nitrogen detection in water, characterized in that: The method comprises reagent A, reagent B and reagent C; the reagent A is a nitrogen determination alloy; the reagent B is a urease extract; the reagent C is a potassium sodium tartrate-sodium hydroxide solution, in which, by weight percentage, the potassium sodium tartrate is 1%-10%, the sodium hydroxide is 0.1%-10%, and the solvent is water; The preparation of the urease extract comprises: The stabilizer and glycerol are mixed, and then soybean powder is added, stirred at room temperature for 0.5-1.5 hours, and then allowed to stand at 0-5°C for 20-25 hours, and the supernatant is taken and filtered, and the obtained filtrate is the urease extract; The mass ratio of the stabilizer, glycerin and soybean powder is 60-100:5-50:10; The stabilizer, based on 250 mL, contains 1-10 g of anhydrous citric acid, 1-10 g of anhydrous disodium hydrogen phosphate and 1-10 g of EDTA-2Na, the solvent is water, and the pH is 5-7.

2. The water sample pretreatment reagent according to claim 1, characterized in that: In the preparation of the urease extract, the solution was stirred at room temperature for 1 hour and then allowed to stand at 0-5° C. for 24 hours.

3. The water sample pretreatment reagent according to claim 1, characterized in that: In the preparation of the urease extract, the mass ratio of the stabilizer, glycerol and soybean powder is 80-100:5-15:

10.

4. The water sample pretreatment reagent according to claim 1, characterized in that: In the preparation of the urease extract, the stabilizer, based on 250 mL, comprises 1-2 g of anhydrous citric acid, 3-6 g of anhydrous disodium hydrogen phosphate and 5-10 g of EDTA-2Na, the solvent is water, and the pH is 5-7.

5. The water sample pretreatment reagent according to claim 1, characterized in that: In the sodium potassium tartrate-sodium hydroxide solution, by weight percentage, the sodium potassium tartrate comprises 3%-5%, the sodium hydroxide comprises 0.1%-5%, and the solvent is water.

6. A water sample pretreatment method for detecting total nitrogen in water using the water sample pretreatment reagent according to any one of claims 1 to 5, characterized in that: include: Take the test water sample, add the reagent A and mix well, then add the reagent B and mix well, carry out enzymatic hydrolysis for 4 to 6 minutes, then add the reagent C, mix well and react for 8 to 12 minutes to obtain the pretreatment solution; Based on 1 mL of the test water sample, the added amounts of reagent A, reagent B and reagent C are 30-60 mg, 100-200 uL and 3-5 mL respectively.

7. A kit for detecting total nitrogen in water, characterized in that: The invention comprises an ammonia nitrogen detection reagent and a water sample pretreatment reagent as claimed in any one of claims 1 to 6.

8. The detection kit according to claim 7, characterized in that The ammonia nitrogen detection reagent is an ammonia nitrogen detection reagent with an ammonia nitrogen detection concentration range of 0-20 mg / L.

9. A method for detecting total nitrogen in water using the detection kit according to claim 7, characterized in that: include: Take the test water sample, add the reagent A and mix, then add the reagent B and mix, enzymatically react for 4-6 minutes, then add the reagent C, mix and react for 8-12 minutes to obtain the pretreatment solution; based on 1 mL of the test water sample, the added amounts of the reagent A, reagent B and reagent C are 30-60 mg, 100-200 uL and 3-5 mL respectively; The obtained pre-treatment liquid is filtered and the filtrate is taken, diluted in proportion and then added with the ammonia nitrogen detection reagent, and the absorbance value at 620 nm is detected after standing for 4 to 6 minutes; Substitute the obtained absorbance value into the standard curve and calculate the total nitrogen concentration in the test water sample.

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